Apolipoprotein (apo) A-IV is a 46 kDa lipid-binding protein initially isolated as a component of intestinally derived lipoproteins. Upon entry into plasma from the intestinal lymphatics, human apoA-IV rapidly dissociates from chylomicrons and either associates with high density lipoproteins or exists as a significant fraction of lipid-free apoA-IV. This raises the possibility that alternate conformational states of apoA-IV may perform distinct functions in lipoprotein metabolism. Indeed, in addition to a potential role in chylomicron assembly, apoA-IV has also been postulated to perform diverse functions including participation in reverse cholesterol transport, inhibition of inflammatory processes, and regulation of food intake. To understand how a single protein can mediate these varied effects requires detailed knowledge of the structure/function relationships of apoA-IV in its various states. The goals of this application are a) to derive all atom molecular models for apoA-IV in both lipid-associated and unassociated states, b) to understand the molecular determinants of apoA-IV lipid affinity, and c) to determine the role of apoA-IV lipid binding on lipoprotein metabolism in vivo. The structural models will be derived using state-of-the-art techniques that combine high resolution mass spectrometry and computerized modeling strategies. The resulting models will be rigorously evaluated with experimental data from limited proteolysis and spectroscopic studies. We also hypothesize that human apoA-IV contains structural features that attenuate its ability to interact with lipids. To test this, we will use mutagenesis strategies to isolate specific regions within apoA-IV that mediate lipid interactions. This information will be used to engineer apoA-IV mutants that are structurally similar to wild-type but vary in their lipid association. Select mutants will be introduced into cell culture and rodent models and the apoA-IV lipid binding affinity will be correlated with chylomicron assembly and catabolism. The information from these studies will be important not only in terms of the function and potential therapeutic use of apoA-IV against cardiovascular disease, but will also be useful for understanding the structure/function of the entire family of exchangeable apolipoproteins.